The structure of the scaffold nucleoporin Nup120 reveals
a new and unexpected domain architecture
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Citation
Leksa, Nina C., Stephen G. Brohawn, and Thomas U. Schwartz.
“The Structure of the Scaffold Nucleoporin Nup120 Reveals a
New and Unexpected Domain Architecture.” Structure 17.8
(2009): 1082–1091.
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http://dx.doi.org/10.1016/j.str.2009.06.003
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Elsevier
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Author Manuscript
Structure. Author manuscript; available in PMC 2010 August 12.
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Published in final edited form as:
Structure. 2009 August 12; 17(8): 1082–1091. doi:10.1016/j.str.2009.06.003.
The structure of the scaffold nucleoporin Nup120 reveals a new
and unexpected domain architecture
Nina C. Leksa1,2, Stephen G. Brohawn1,2, and Thomas U. Schwartz1,*
1Department of Biology, Massachusetts Institute of Technology 77 Massachusetts Avenue,
Cambridge, MA 02139, USA
SUMMARY
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Nucleocytoplasmic transport is mediated by nuclear pore complexes (NPCs), enormous protein
assemblies residing in circular openings in the nuclear envelope. The NPC is modular, with transient
and stable components. The stable core is essentially built from two multiprotein complexes, the Yshaped heptameric Nup84 complex and the Nic96 complex, arranged around an eightfold axis. We
present the crystal structure of Nup1201-757, one of the two short arms of the Y-shaped Nup84
complex. The protein adopts a compact oval shape built around a novel bipartite α-helical domain
intimately integrated with a β-propeller domain. The domain arrangement is substantially different
from the Nup85•Seh1 complex, which forms the other short arm of the Y. With the data presented
here we establish that all three branches of the Y-shaped Nup84 complex are tightly connected by
helical interactions and that the β-propellers likely form interaction site(s) to neighboring complexes.
INTRODUCTION
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The main feature that distinguishes eukaryotes from prokaryotes is the confinement of the
genetic material into a membrane-enveloped nucleus. Since gene transcription and mRNA
processing occur inside the nucleus while protein translation is restricted to the cytoplasm,
transport across the double-layered nuclear envelope (NE) is essential for cellular homeostasis.
The exchange of all molecules, including ions, proteins, and RNAs is facilitated exclusively
by nuclear pore complexes (NPCs) (D'Angelo and Hetzer, 2008; Lim et al., 2008; Tran and
Wente, 2006; Weis, 2003). NPCs are large protein assemblies of 40-60 MDa that are embedded
in the nuclear envelope and exhibit an 8-fold rotational symmetry around a central axis in
addition to an imperfect two-fold symmetry across the plane of the NE (Beck et al., 2007;
Stoffler et al., 2003). Composed of multiple copies of ∼ 30 proteins, termed nucleoporins
(nups), the NPC has an outer diameter of ∼100 nm while the central channel measures ∼40 nm
in width. Transmembrane nups directly connect the NPC to the NE, while the phenylalanineglycine (FG) repeat-containing Nups line the interior of the pore. These FG-filaments mediate
nucleocytoplasmic transport of cargo molecules across the NE. FG-filament bearing nups are
anchored to the NPC scaffold built from architectural nucleoporins arranged in two large
© 2009 Elsevier Inc. All rights reserved.
*Correspondence: tus@mit.edu.
2These authors contributed equally to this work
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ACCESSION NUMBER
The atomic coordinates of the Nup120 structure have been deposited in the Protein Data Bank (PDB) with accession number 3HXR.
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multiprotein complexes that form a membrane-proximal layer. The scaffold structure is very
stable and undergoes virtually no turnover in the quiescent cell (D'Angelo et al., 2009), while
many other nucleoporins have variable dwell times at the NPC (Rabut et al., 2004). In
consequence, the NPC is a highly modular structure (Schwartz, 2005). Understanding the
structure of the NPC therefore depends upon elucidating its basic scaffold.
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The two essential architectural building blocks of the NPC are the Nup84 subcomplex and the
Nic96 subcomplex. The components of the Nic96 subcomplex likely include Nic96, Nup53/59,
Nup157/170, Nup188 and Nup192 (yeast nomenclature), as inferred from coimmunoprecipations (co-IPs) (Alber et al., 2007; Hawryluk-Gara et al., 2008; Marelli et al.,
1998; Onischenko et al., 2009) and yeast-two-hybrid screens (Wang et al., 2009; Yu et al.,
2008). Judged by immunolabeling, the Nic96 subcomplex might form a central ring within the
NPC sandwiched between peripheral rings formed by Nup84 subcomplexes (Alber et al.,
2007). In comparison to the Nic96 subcomplex, the Nup84 subcomplex is substantially better
understood. It has 7 universally conserved members (yeastNup84/humanNup107, yNup85/
hNup75, yNup120/hNup160, Nup133, yNup145C/hNup96, Sec13, and Seh1) and three
additional members (Nup37, Nup43, and ELYS/Mel-28) to date found mainly in metazoa
(Cronshaw et al., 2002; Gillespie et al., 2007; Loiodice et al., 2004; Rasala et al., 2006). In the
fungus Aspergillus nidulans, distant Nup37 and ELYS orthologs have been described recently
(Liu et al., 2009). The heptameric core Nup84 complex assembles tightly as shown by co-IPs
and in vitro assembly (Harel et al., 2003; Lutzmann et al., 2002; Siniossoglou et al., 2000;
Walther et al., 2003). Negatively-stained electronmicrographs of the assembled Nup84
complex reveal a branched Y-shaped structure, with two short arms and a kinked stalk
connected at a central hub (Lutzmann et al., 2002).
Crystallographic analysis of the Y-complex has progressed quickly. The kinked stalk ends with
a flexibly attached β-propeller domain (Berke et al., 2004) at the N-terminus of Nup133
followed by an irregular C-terminal helical stack domain that connects end-to-end to Nup84
(Boehmer et al., 2008); Whittle, unpublished). The Nup84•Nup133 interface defines at least
one kink in the stalk. The opposite end of Nup84 links to Nup145C (Brohawn et al., 2008).
Nup145C•Sec13 (Hsia et al., 2007) resides proximal to the hub (Lutzmann et al., 2002).
Nup85•Seh1 forms one of the two short arms of the Y-shaped complex (Brohawn et al.,
2008; Debler et al., 2008). Nup84, Nup85, and Nup145C are structurally related (Brohawn et
al., 2008), despite very low sequence conservation, as are the β-propeller proteins Seh1 and
Sec13.
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Nup120 is the last remaining Y-complex component without structural information. Here we
report the 3.0 Å crystal structure of Nup120 (residues 1-757 of 1037), which reveals a compact
and rigid structure composed of an N-terminal β-propeller domain tightly integrated into a
novel bipartite α-helical domain. Our structure largely defines the second short arm of the Ycomplex. Comparison with other members of the Y-complex, phylogenetic analysis, in vitro
binding experiments, and in vivo localization data suggest a role for Nup120 consistent with
our lattice-like model of the NPC.
RESULTS
Structure Determination
After systematic C-terminal truncation, a stable fragment comprising most of Nup120 (residues
1-757 of 1037 total) from S. cerevisiae was recombinantly expressed in E. coli and purified.
The protein is a monomer in solution (data not shown). Native protein readily crystallized and
selenomethionine derivatized crystals were obtained after microseeding with native crystals.
Though both crystal forms were optically identical, the selenomethionine crystals diffracted
better and were used exclusively in structural analysis. The structure of Nup120 was solved
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with one molecule per asymmetric unit by single-wavelength anomalous dispersion (SAD) on
very strong Se-Peak data (all 9 Se sites are well ordered). The model is complete except for 27
residues at the C-terminus and 7 flexible loops (out of 43 total loops) and was refined to
Rwork / Rfree of 24.4 % / 29.9 % (Table 1).
Crystal Structure of Nup120
Nup120 folds into a continuous, prolate disk with overall dimensions of 90 Å × 55 Å × 35 Å.
One half of the structure is formed by an N-terminal β-propeller domain that is intimately
connected to a compact central domain built from two closely packed α-helical segments
(Figure 1). Overall, the structure is better resolved in the α-helical segment than the β-propeller,
likely a result of a paucity of packing contacts involving the latter. The β-propeller of Nup120
contains 7 consecutive blades that fan out from a central axis. The blades are formed by a βsheet of 4 consecutive antiparallel strands, labeled A-D. Blade 7 is built from the very Nterminus of the polypeptide chain forming strand 7D and joining strands 7A-C to close the
propeller in a velcro-like closure commonly observed in β-propeller domains (Chaudhuri et
al., 2008). Blade 1 is 5-stranded, with strand 7D extending to form the additional strand 1E
before connecting to strand 1A (Figure 1E). Blade 3 is somewhat unusual in that the outermost
strand 3D is only loosely connected to strand 3C with a hydrogen-bonding network hardly
visible in our structure and the sequence could only be tentatively assigned for strand 3D
residues 204-216.
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The α-helical domain that forms the second half of the molecule is constructed in a unique
discontinuous manner. In total the domain contains 15 helices, labeled α1-α15. The first 4
helices form a compact bundle and are inserted between blades 6 and 7 of the β-propeller. The
remaining 11 helices are C-terminal to the β-propeller and pack tightly against the 4-helix
bundle to form one compact entity. The arrangement of the helices is highly irregular. The
most prominent feature of the domain are two long helices, α11 and α12, which pack against
each other and form a central stalk, defining the long axis of the domain. Helices α5-α9 wrap
up and around this element, with helices α6/α7 and α8/α9 arranged in two stacked braces
oriented perpendicular to the stalk. Helices α1, and α13-α15 meander back down and around
the other side to bury most of the hydrophobic stalk. The remaining surface area of the two
central helices is closed by the 4-helix insertion bundle. Taken as a whole, the structure of
Nup1201-757 consists of a bipartite helical domain that is interrupted by a β-propeller.
The Main Crystal Contact is Formed by a Domain Swap
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Other than a collection of spurious small contacts crystal packing is mainly achieved by a
domain swap of the terminal helices α15 and α15′ exchanging between two neighboring
molecules (Figure 2A). The interface measures 1355 Å2, is entirely hydrophobic and highly
complementary (Figure 2B). Domain swaps are regularly found in crystals (Liu and Eisenberg,
2002) and, as stated above, we do not observe dimerization of Nup120 in solution. We cannot
rule out the possibility that the interface is physiologically relevant; sterically the domain swap
is conceivable in the context of the entire molecule including the C-terminal 280 residues
omitted in our construct. It is however more likely that the exposed hydrophobic patch is
artificially generated by the truncation of the domain, since we also do not observe particularly
high sequence conservation within helix α15. We speculate that in vivo the patch likely
accommodates one of the additional helices from the C-terminal domain, or alternatively, is
involved in interaction with a neighboring molecule. Whether the C-terminal domain is rigidly
or flexibly tethered to Nup1201-757 is an open question.
Conservation of Nup120 and Comparison to the Human Ortholog Nup160
Overall, sequence conservation between Nup120 orthologs is weak as is typically observed in
scaffold nucleoporins (Brohawn et al., 2008; Jeudy and Schwartz, 2007). Most of the betterStructure. Author manuscript; available in PMC 2010 August 12.
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conserved residues are buried in the hydrophobic core of the protein and are involved in
maintaining the structural integrity of the protein. On the protein surface we find few conserved
patches (Figure 3A). Most distinct is an area on the edge of the β-propeller, corresponding to
the outer strands of blade 3 and the loop leading into blade 4. The conserved sequence begins
in the 3BC loop and continues into strand 3C itself. Although generally buried in canonical
β-propellers, here strand 3C is quite exposed. This is probably the result of weaker interactions
with strand 3D, which is flanked by two large loops and peels away from the core of the
propeller. Additional conserved residues are spotted around this area, creating a relatively large
conserved patch. The potential significance of this observation is discussed below.
We analyzed the charge distribution on the surface of Nup120 (Figure 3B). Since Nup120 is
part of the scaffold structure of the NPC, we asked whether it may be possible that it directly
juxtaposes the pore membrane. This would also be consistent with a membrane-curvature
sensing ALPS motif, predicted in helix α5-6 of Nup120 (Drin et al., 2007). The surface charge
of Nup1201-757, however, is fairly mixed without conserved positive patches that might suggest
direct membrane interaction. The ALPS motif is embedded in the structure and it is rather
unlikely that it would swing out and insert in the membrane. Thus we suggest that
Nup1201-757 does not directly touch the nuclear membrane.
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Structure-guided sequence comparison of Nup120 and its human ortholog Nup160 strongly
suggests that both proteins adopt the same unique fold despite a low sequence identity of ∼10
%. Both non-canonical characteristics of Nup120 (the helical insertion between blades 6 and
7 of the N-terminal β-propeller and the long central stalk helices forming the hydrophobic core
of the central domain) are clearly conserved in Nup160. The 279 additional residues of Nup160
are dispersed over several regions and mostly correspond to different loop lengths connecting
α-helices and β-strands. Of note, the C-terminal domain of Nup160, which is not present in the
Nup120 crystal structure described here, has 5 additional predicted helices, possibly indicating
a vertebrate-specific extension. Despite these differences, the Nup120 crystal structure is likely
generally representative of all Nup120/Nup160 orthologs.
The C terminus of Nup120 Directly Binds Nup145C and Nup85
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We sought to map the interaction of Nup120 with its direct binding partners in the Y-complex,
Nup145C and Nup85. In a gel filtration assay, we tested for the formation of a pentameric
Sec13•Nup145C•Nup120•Nup85•Seh1 complex (Figure 4). Incubating Nup120766-1037 or
Nup1201-757 with both Nup145C• Sec13 and Nup85•Seh1 resulted in complex formation only
for the C-terminal Nup120 domain, but not for the crystal construct. In combination with
previous interaction mapping experiments (Brohawn et al., 2008), we conclude that the helical
tails of the ACE1 domains of both Nup145C and Nup85 each interact directly with the helical
Nup120766-1037. This positions the C-terminus of Nup120 at the center of the hub of the Ycomplex.
Without its C-terminal domain Nup120 does not properly localize to the NPC
Having established that Nup120766-1037 is sufficient to bind both Nup145C• Sec13 and
Nup85•Seh1 in vitro, we sought to examine the integration determinants of Nup120 into the
NPC in vivo. NUP120 is not essential in yeast but nup120 Δ cells exhibit a pore clustering
phenotype (Aitchison et al., 1995; Heath et al., 1995) that is reminiscent of but less severe than
the pore clustering observed for other scaffold nucleoporins including Nup84 and Nup133 (Li
et al., 1995; Pemberton et al., 1995; Siniossoglou et al., 1996). We genomically GFP-tagged
full length Nup120 and replaced the C-terminal 280 residues of genomic Nup120 with an in
frame GFP-tag to create strains expressing Nup120-GFP or Nup1201-757-GFP in a BY4741
background and examined the localization of the proteins via immunofluorescence (Figure 5).
Nup120-GFP properly localizes to the NPC and shows typical nuclear rim staining,
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superimposing well with mAb414-staining of FG-Nups (Aris and Blobel, 1989).
Nup1201-757-GFP, on the other hand, does not properly localize to the nuclear envelope and
shows staining throughout the cell. This result is consistent with our in vitro data and suggests
that the integration into the Y-complex is important for proper localization of Nup120.
Nup120 is Topologically Different from other Scaffold Nucleoporins
A recent surge in the X-ray crystallographic analysis of components of the NPC has greatly
increased the repertoire of available structures of nucleoporins constituting the structural
scaffold of the NPC. These structures (including those of Nic96 (Jeudy and Schwartz, 2007;
Schrader et al., 2008), Nup133-NTD (Berke et al., 2004), Nup133•Nup107 interaction complex
(Boehmer et al., 2008), Nup145C•Sec13 (Hsia et al., 2007), and Nup85•Seh1 (Brohawn et al.,
2008; Debler et al., 2008) as well as associated biochemical experiments, have led to a deeper
and broader understanding of how the scaffold of the NPC is assembled from its constituent
parts.
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The structural subunits of the NPC were initially predicted to be composed of simple
combinations of regular α-helical solenoids and β-propellers (Devos et al., 2006). Experimental
data now allows to specify these broad classifications, which should help to more specifically
address the ancestry of the NPC. Both Sec13 and Seh1 form open, 6-bladed propellers that are
completed in trans by the N-terminal insertion blades of their binding partners Nup145C and
Nup85, respectively. Furthermore, helical nucleoporins Nic96, Nup145C, Nup85, and Nup84
are built around a common and distinct ancestral coatomer element (ACE1) shared with Sec31
of the outer coat of COPII vesicles (Brohawn et al., 2008). In ACE1 proteins, a specific Nterminal elaboration is followed by a tripartite helical domain composed of a trunk, a crown
and a tail element. The ∼ 30 helices within ACE1 follow a J-like pattern, zig-zagging up on
one side of the trunk, making a U-turn within the crown domain, and then following down on
the opposite side of the trunk (Figure 6A, right panel). The tail domain is often attached with
modest flexibility to the trunk and is missing in most crystal constructs. In the case of Nup145C
and Nup85 the N-terminal elaborations are the aforementioned insertion blades that bind to
Sec13 and Seh1. Nup145C•Sec13 and Nup85•Seh1 heterodimers form the two proximal
segments of the Y-shaped complex and are tethered together by Nup120 (Brohawn et al.,
2008).
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Based on structure predictions and its overall size, it was reasonable to suggest that Nup120
may take on a structure similar to Nup145C•Sec13 and Nup85•Seh1, with the only major
difference being that the β-propeller and the α-helical domains are fused into one polypeptide
chain. However, comparison between the structure of Nup120 and the Nup85•Seh1
heterodimer reveals a marked difference in topology (Figure 6A). Whereas the ACE1
architecture of Nup85 forms an elongated α-helical domain, the central α-helical domain of
Nup120 is nearly as wide as it is long, forming an almost globular structure. The ACE1 trunk
module covers the bottom face of the Seh1 β-propeller, while in Nup120 the helical domain is
attached to and integrated into an edge of the β-propeller. Further, the ACE1•β-propeller
interaction is accomplished by the addition of an insertion blade N-terminal to ACE1, while
in Nup120 the β-propeller domain inserts a 4-helix bundle into the central α-helical domain.
This helical insertion fits snugly into a pocket formed by helices α5-α7 and α11-α13 and
creating an interface of nearly 600 Å2 (Figure 6B).
The extensive interaction between the β-propeller and α-helical domain of Nup120 creates a
large, rigid interface of 2175 Å2. In contrast, the largest contact area between ACE1 and its
β-propeller partner is at the insertion blade/β-propeller interface. Additional contact areas in
ACE1•β-propeller complexes are smaller in comparison to the corresponding interfaces in
Nup120 and, importantly, far less hydrophobic. Thus, for the ACE1•β-propeller assembly one
has to consider substantial flexibility about the interaction joint, while the Nup120 structure
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presented here is very likely inflexible. Not only does the structure of Nup120 significantly
differ from the ACE1•β-propeller heterodimers, but additional emerging evidence suggests
that it also lacks similarity to Nup170 and Nup133, the two other scaffolding nucleoporins of
similar size and domain composition with an N-terminal β-propeller followed by an α-helical
domain (Whittle & Schwartz, manuscript in preparation).
DISCUSSION
Here we report the crystal structure of Nup120, a large, universally conserved architectural
nucleoporin. This structure adds substantially to the growing inventory of crystallographically
characterized nucleoporins. As a result of these studies, we learn that the NPC is constructed
from nucleoporins with a limited set of domain architectures. While other α-helical and βpropeller domains of scaffold nucleoporins fall into distinct classes, likely pointing to gene
duplication in the early evolution of the NPC, the Nup120 architecture appears to be quite
distinct. A search for structurally related proteins fails in detecting similarity beyond the
isolated β-propeller scaffold or the arrangement of more than 6 α-helices. Within the list of
crystallographically uncharacterized nucleoporins, none is likely to match the Nup120
structure closely.
Nup120 in the Context of the NPC Scaffold
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Nup120 forms one of the two short arms of the universally conserved, 0.6 MDa Y-complex,
the essential building block of the NPC scaffold. The assembly of the Y-complex from its 7
members is fairly well understood and has been studied using many different techniques. All
of these studies profit from generally very high affinities observed between the interacting
proteins within the Y, which generated largely consistent co-immunoprecipitation and yeast
two-hybrid results and facilitated the crystallization of several complex crystal structures.
While there is general agreement on the overall topology of the NPC, as determined by electron
microscopic techniques, different models for the assembly of the NPC structural scaffold and
the integration of the Y-complex are being discussed, as more detailed information is becoming
available.
Based on a combination of computational, structural, biochemical, and in vivo experiments, a
model was proposed where the Y-complex is positioned in two 8-membered rings located at
the periphery of the NPC sandwiching two equally wide rings composed of
Nup157/170,Nup188 and Nup192 in between (Alber et al., 2007). One exiting aspect of the
combinatorial approach is, that going forward to a higher resolution it may allow the integration
of crystallographic data as well, in which case it could come close to a detailed molecular
description of the NPC.
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Blobel and coworkers proposed a concentric cylinder model based on crystal-packing
interactions where four 8-membered rings of the Y-complex are stacked and placed directly
adjacent and in contact to the curved membrane (Debler et al., 2008; Hsia et al., 2007). Further,
Nup85•Seh1 and Nup145C•Sec13 are both supposed to form heterooctameric fence poles
spanning the NPC vertically, thereby connecting the 4 stacked rings. Nup157/170, Nup188,
Nup192 and Nic96 are suggested to form a second inner layer bridging to a third layer
composed of FG-nups. With a Y-complex scaffold twice the mass of the computer-generated
model, the concentric cylinder model generates a densely packed NPC coat.
In contrast to the concentric cylinder model, we proposed a lattice-like model for the NPC,
extrapolated from the assembly of COPII vesicle coats and substantiated by the structure and
assembly principles of core components of the NPC scaffold (Brohawn et al., 2008; Brohawn
and Schwartz, 2009). We propose the Y-complex does not directly coat the pore membrane
(in analogy to the COPII outer coat), but is anchored by another set of proteins, likely involving
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the essential transmembrane nucleoporin Ndc1 and/or its direct binding partners (Onischenko
et al., 2009). It is of interest to discuss this issue in respect to the membrane-inserting ALPS
motif that was experimentally characterized within a loop structure in hNup133-NTD and that
was predicted to occur as well in yNup85 and yNup120 (Drin et al., 2007). Based on the
structural data now available on both yNup85 and yNup120, it appears unlikely that the
predicted ALPS motif in both proteins is functional in membrane-binding since neither is in
an exposed region of the protein, or is likely to become exposed. This is in contrast to the ALPS
motif in hNup133-NTD, where it is well exposed in the crystal structure, and also highly
conserved in metazoa (Berke et al., 2004). Taking all the available data together, it appears
more reasonable to suggest a specific function for the ALPS motif in metazoan Nup133 rather
than a general function in anchoring of the NPC to the pore membrane. Since Nup133-ALPS
is only poorly conserved in yeast, it is tempting to speculate that it may have a specific role in
NPC assembly in open mitosis (Güttinger et al., 2009). We predict that the lattice scaffold of
the NPC is built from edge and vertex elements, following similar assembly principles as
established for COPII. However, in the absence of definitive inter-subcomplex interaction data,
any detailed NPC assembly model is still premature and has to be interpreted cautiously.
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The fact that inter-subunit interactions are still obscure suggests that these interactions are
rather weak and hard to establish. Each short arm of the Y-complex contains one β-propeller
domain, while the stalk contains two (Figure 1). For the assembly of the extensions of the Y,
direct interactions between the α-helical domains is essential, however this does not exclude
the participation of the β-propellers. It is reasonable to suggest, that the β-propellers are prime
candidates for the elusive inter-subcomplex contacts. The vertices of the outer coat of COPII
vesicles are assembled exclusively via β-propeller interactions, which have still only been
inferred by fitting crystal structures into EM maps (Fath et al., 2007;Stagg et al., 2008). βpropellers make excellent protein-protein interfaces due to their inherent ability to pair with a
binding partner in multiple modes. Binding to peptides via the face of the β-propeller is well
known (Jawad and Paoli, 2002). Additionally, each blade exposes on its edge (typically on
strand D) a stretch of ∼ 6-8 residues available for intermolecular β-sheet formation, which can
be likened to one half of a zipper. In Nup120, 5 of the 7 blades are exposed this way, two are
buried in the hydrophobic core shared with the attached α-helical domain. In addition to these
interactions being relatively weak, another inherent difficulty in identifying them is that they
are likely very poorly conserved at the sequence level because the contacts are mediated via
the backbone rather than side chains. Based on the available data, it is conceivable that the
Nup120 β-propeller is involved in inter-Y contacts. It is also possible that it is used to bridge
to the Nic96 complex, but we can also not exclude that it may be an anchor for dynamic
nucleoporins or other accessory proteins. The relatively mild nup120Δ phenotype (Figure 5)
compared to nup133Δ or nup84Δ and the behavior of Nup1201-757-GFP suggests that if the
Nup120 β-propeller has an integral role in the NPC scaffold, it is either redundant or can be
functionally replaced by another nucleoporin.
In summary, we show that Nup120 adopts a unique architecture to build one of the two arms
of the multimeric Y-shaped complex, the linchpin of the NPC scaffold. The atomic structure
of the universally conserved heptameric core of the Y complex is now nearing completion.
With reliable data on inter-subcomplex contacts the construction of a basic NPC architecture
is within reach in the close future.
EXPERIMENTAL PROCEDURES
Protein Expression and Purification
Nup120 from S. cerevisiae (residues 1-757 of 1037) was expressed at 18°C in E. coli strain
BL21(DE3)-RIL as a 6xHis N-terminal fusion protein from a pET-Duet-derived plasmid. Cells
were pelleted and resuspended in lysis buffer (50mM potassium phosphate pH 8.0, 500mM
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NaCl, 40mM imidazole, 5mM β-mercaptoethanol). Cells were lysed using a french press and
the clear lysate incubated in batch with Ni-affinity resin. After washing the resin in batch with
lysis buffer, the protein was eluted with lysis buffer containing 250mM imidazole. After
cleavage of the purification tag, Nup120 was subjected to size exclusion chromatography on
Superdex S200 equilibrated in 10mM Tris/HCl pH 8.0, 150mM NaCl, 0.1mM EDTA, and
1mM DTT. Nup120 eluted as a monomer of 88 kDa. Selenomethionine-derivatized protein
was prepared as previously described (Brohawn et al., 2008) and Nup120-SeMet was purified
identically to the native version.
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Full length Nup85 in complex with Seh1 and a single chain version of full-length Nup145C in
complex with Sec13 from S.cerevisiae were cloned as described (Brohawn et al., 2008),
purified as for Nup120 (residues 1-757), and are referred to in the text as Nup85•Seh1 and
Nup145C•Sec13. The C-terminal helical domain of Nup120 (residues 766-1037) was
generated from a full length Nup120 construct by PCR. A 5-protein complex of Nup120
(residues 766-1037), Nup85•Seh1, and Nup145C•Sec13 was prepared by co-expression of a
trimeric complex of Nup120 (residues 766-1037)•Nup85•Seh1 (Brohawn et al., 2008) and the
single chain version of Nup145C•Sec13 in BL21(DE3)-(RIL) cells and was purified as for
Nup120 (residues 1-757). The Ni-NTA elution was pooled, digested with human rhinovirus
3C to remove fusion tags, and subjected to size exclusion chromatography using a Superdex
S200 26/60 column equilibrated in 10mM Tris/HCl pH 8.0, 250mM NaCl, 1mM DTT, and
0.1mM EDTA.
Protein Crystallization
Nup120 concentrated to 20 mg/ml was crystallized in 15% (w/v) PEG 3350, and 0.1M Tris/
HCl pH 7.5, 0.2M KSCN by the hanging drop vapor diffusion method at 18°C in 2μl drops.
Crystals grew within 3-6 days forming rhomboid prisms with dimensions of 60μm × 60μm ×
20μm. The selenomethionine derivative crystallized in the same condition, while the highest
quality crystals were obtained by microseeding with native crystals. Both native and derivative
crystals were cryo-protected by serial transfer of the crystals into reservoir solutions
supplemented with increasing amounts of PEG200 (10%-25% (v/v), 5% steps) before flash
freezing in liquid nitrogen. Both native and derivative protein crystallized in space group
P21212 with one molecule per asymmetric unit. Data was collected at beamline 24ID-C at
Argonne National Laboratory.
Structure Determination
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Although the native crystals were larger and optically superior, the selenomethioninederivatized crystals diffracted significantly better and were exclusively used for data analysis.
A complete dataset was collected at the Se-Peak wavelength and data reduction was carried
out using the HKL2000 package (Otwinowski and Minor, 1997). All 9 selenium sites were
found using SHELXD (Sheldrick, 2008). After refinement of the Se positions and density
modification with SHARP, an adequate experimental electron density map was obtained,
allowing for the assignment and building of the majority of the structure. Sequence assignment
was aided by using the selenium positions as markers. Model building was done with Coot
(Emsley and Cowtan, 2004) and refinement was carried out using the PHENIX suite (Adams
et al., 2002). The model is complete except for residues 31-52, 188-200, 303-313, and 731-757
for which only spurious electron density was observed. Blades 3 and 4 of the β-propeller have
the highest temperature factors and are not as well packed as the remainder of the molecule.
Sequence assignment in this region, particularly in strand 3D residues 204-216, is tentative.
Analytical Size Exclusion Chromatography
For Nup120 (residues 1-757), Nup145C•Sec13, and Nup85•Seh1 binding experiments,
equimolar amounts of each component were incubated alone or in combination for 30 minutes
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at 4°C in binding buffer (10mM Tris/HCl pH 8.0, 250mM NaCl, 1mM DTT, 0.1mM EDTA).
Reactions were injected onto a Superdex S200 hr10/300 column (GE Healthcare) equilibrated
in binding buffer, and run at a flow rate of 0.8 ml/min (Figure 4A).
Yeast Strain Construction
Deletion strains were taken from the Yeast Deletion Consortium (Winzeler et al., 1999), Cterminal GFP-tagging was done by homologous recombination in a BY4741 background, using
pFA6a-GFP(S65T)-kanMX6 as template for C-terminal modifications (Longtine et al.,
1998). Strains were selected on G418 plates (200 μg/ml) and verified by PCR.
Fluorescence Microscopy
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Strains were grown overnight in YPD (1% yeast extract, 2% yeast peptone, 2% glucose) at 30°
C, diluted 20-fold into fresh YPD, and grown for 4-5 hours at 30°C to OD600 ∼0.5. Cells were
harvested by centrifugation, fixed for 3 minutes in 3.7% formaldehyde/0.1M potassium
phosphate pH 6.5, and prepared for immunofluorescence as previously described (Kilmartin
and Adams, 1984). Samples were incubated with mAb414 (abcam, 1:1000) alone or in
combination with goat anti-GFP (1:500) for 90 minutes at room temperature. Bound antibodies
were detected by incubation with Cy5-conjugated anti-mouse (Jackson Labs, 1:500) alone or
in combination with Cy2-conjugated donkey anti-goat (Jackson Labs, 1:200) for 45 minutes
at room temperature. DNA was stained with 0.05 μg/ml 4′,6-diamidino-2-phenylindole
(Sigma-Aldrich) and samples were mounted for imaging in 1 mg/ml p-phenylenediamine and
90% glycerol. Fluorescence microscopy was performed on a Zeiss AxioImager.Z1 microscope
and images were taken with a Zeiss AxioCam HRm camera.
ACKNOWLEDGEMENTS
We thank staff at the NE-CAT beamline 24 at Argonne National Laboratory for excellent assistance with data
collection; Iain Cheeseman for providing antibodies; Eric Spear for advice on in vivo experiments; Dennis Kim for
use of his fluorescence microscope. Supported by NIH Grant GM77537 (T.U.S.), a Pew Scholar Award (T.U.S.), a
Koch Fellowship Award (S.G.B.) and a Vertex Scholarship (S.G.B.).
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Figure 1. Overall Topology of Nup120
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(A) Current model of the Y-shaped Nup84 subcomplex. The relative position of Nup120 is
highlighted. (B) Schematic of full-length Nup120 from S. cerevisiae. Residues that form the
β-propeller are colored blue, those that form the α-helical domain are purple, and those not
present in the crystallized construct are in gray. (C, D) The overall topology of Nup120
(residues 1-757 of 1037) is shown in two views rotated by 90°. The structure is gradient-colored
from blue to white to magenta from N- to C-terminus. At its N-terminus, Nup120 forms a 7bladed β-propeller. A 4-helix bundle (α1-α4) between blades 6 and 7 packs against the
remainder of the helical domain (α5-α15), composed of helices wrapping around a central
hydrophobic stalk of the two long helices α11 and α12. Unstructured loops absent from the
final model are shown in gray. (E) A topological diagram of the Nup120 structure is shown,
illustrating the 4-helix insertion between blades 6 and 7 of the propeller as well as the two
central helices of the helical domain.
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Figure 2. Crystal Contacts between Two Symmetry-Related Molecules
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(A) One molecule of Nup120 in blue, and its symmetry mate, related by a 2-fold rotation, in
orange. The β-propellers are at opposite ends while the helical domains engage in a putative
domain swap between helices α15 of both molecules. (B) Close-up of the domain-swapped
region, illustrating the hydrophobic nature of helix α15 and the surrounding pocket
(hydrophobic residues are shown in white). In monomeric Nup120, helix α15 likely folds under
(arrow) and occupies the position taken by helix α15′ (orange) of the symmetry-related
molecule in the crystal.
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Figure 3. Surface Conservation and Electrostatics of Nup120
(A) Surface conservation of Nup120 is shown from three different views. To illustrate the
conservation of residues on the surface of Nup120, a multiple sequence alignment sampling
the phylogenetic tree of budding yeasts was generated and mapped onto the surface, colored
from white (not conserved) to orange (highly conserved). The view in the middle panel
corresponds to the view shown in Figure 1C. A patch of highly conserved residues is apparent
on the outer face of the propeller domain of Nup120. (B) The electrostatic surface potential of
Nup120 is shown in the same views as in (A) and is colored from red (−10 kT/e) to blue (+10
kT/e).
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Figure 4. The C-Terminus of Nup120 Is Necessary for Binding Nup85 •Seh1 and Nup145C•Sec13
(A) Nup85•Seh1 (red), Nup145C•Sec13 (gray), and Nup1201-757 (green) were run individually
and in combination (blue) on a Superdex S200 10/300 gelfiltration column. (B) Nup85•Seh1,
Nup145C•Sec13, and Nup120766-1037 were incubated together and run on Superdex S200
26/60 and eluted in a single peak. (C) Fractions from the gel filtration experiment in B were
analyzed by SDS-Page. Co-migration of Nup85•Seh1, Nup145C•Sec13, and Nup120766-1037
indicates that the C-terminus of Nup120 is necessary for the formation of the pentameric
complex that comprises the hub of the Y-shaped complex.
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Figure 5. Nup1201-757 Does Not Localize to the Nuclear Envelope
(Aa-Ad) Nup120-GFP is targeted to the nuclear envelope, as confirmed by co-localization with
mAb414 (staining FG-Nups), while Nup1201-757-GFP (Ba-Bd) is distributed throughout the
cell. Mislocalization indicates that the C-terminus of Nup120 is necessary for proper
recruitment to the NPC. Nup120Δ, nup133Δ, and nup84Δ cells (in the same BY4741 strain
background) are shown for comparison (Cb-Cd, Db-Dd, Eb-Ed). Nuclear rim was visualized
using mAb414, GFP-tagged Nup120 using goat α-GFP, and DNA using DAPI. Merged images
are shown on the right.
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Figure 6. Nup120 is Composed of a combined β-Propeller - α-Helical Domain distinct from
ACE1•β-Propeller
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(A) The overall architectures of Nup120 and the ACE1 motif of Nup85•Seh1 are distinctly
different. Nup120 is characterized by a bipartite helical domain (blue to white from N- to Cterminus) that is interrupted by a β-propeller domain (gray). The Nup85 ACE1 motif is
characterized by an elongated helical stack (colored blue to white from N- to C-terminus) that
makes a U-turn in the crown domain of the molecule. At its N-terminus, Nup85 inserts a blade
(in red) into the open, 6-bladed Seh1 β-propeller. In contrast, the β-propeller of Nup120
contributes a helical insertion bundle (red) to the helical domain. The view of Nup120 is the
same as that in Fig. 3B. (B) Surface representations of intact Nup120 are shown on the left,
while on the right the three modules of Nup120 – the propeller, the helical insertion, and the
helical domain – are shown pulled apart to illustrate the buried surface areas in between.
Interacting surfaces between the propeller and the insertion bundle are outlined in green,
between the propeller and the helical domain in yellow, and between the insertion bundle and
the helical domain in orange. The molecule is N-to-C gradient-colored from blue-to-white-tomagenta.
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Table 1
Data Collection and Refinement Statistics
Data Set
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Nup1201-757
SeMet
Data Collection
Space group
P21212
Cell dimensions
a, b, c (Å)
114.6, 153.7, 53.0
α, β, γ (°)
90.0, 90.0, 90.0
No. of unique reflections
Resolution (Å)
35895
50 - 3.0 (3.1-3.0)
a (%)
Rsym
5.1
Completeness (%)
99.8 (99.6)
Redundancy
3.0 (3.0)
I/σ
20.4 (1.8)
Refinement
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Resolution (Å)
50 - 3.0
No. of unique reflections
35841
No. atoms
Protein
5305
Water
0
Rwork
b
24.4
Rfreec
29.9
RMSD Bond lengths (Å)
0.017
RMSD Bond angles (°)
1.915
B factor (Å )
2
β-propeller
114
α-helical insertion bundle
89
α-helical domain
83
Ramachandran plot (%)d
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favored/allowed/outliers
93.61/5.3/1.09
a
Rsym = |Ii − Ii|/Ii, where Ii is the intensity of the ith observation and Ii is the mean intensity of the reflection
b
Rwork = (||Fobs| − |Fcalc||/|Fobs|)
c
Rfree = R value for a randomly selected subset (5%) of the data that were not used for minimization of the crystallographic residual
d
Calculated using MolProbity
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